Edit-R All-in-one lentiviral sgRNA negative controls

ゲノム編集
ゲノム編集試薬
Edit-R All-in-one lentiviral sgRNA negative controls

Edit-R All-in-one lentiviral sgRNA negative controls

バイオインフォマティクスに基づき、ヒトまたはマウスのゲノムのどの遺伝子も標的にしないように設計および検証されたall-in-one non-targetingコンストラクト

non-targetingコントロールは、実験ベースラインを確立し、配列特異的な生物学的効果と非特異的効果を区別します。製品形態は、高力価の精製レンチウイルス粒子またはグリセロールストックフォーマットをご用意しています。

AAVS1/Rosa26 cutting controls are designed to target regions of the genome that do not result in functional knockout of a protein or known phenotypic changes. These regions are sometimes known as safe harbor regions. These guides are validated for mismatch detection assays and are recommended for screening applications as an additional control that results in Cas9 cutting without a phenotypic readout.

Non-targeting controls are recommended as negative controls for experiments using synthetic sgRNA. All Edit-R non-targeting controls are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in the human or mouse genomes. Changes in viability or gene expression levels in cells treated with these controls likely reflect a baseline cellular response that can be compared to the levels in cells treated with target-specific sgRNAs.

Edit-R all-in-oneレンチウイルスシステムを用いた場合の編集頻度（TIDE）

Edit-R All-in-one editing data

JurkatまたはK562細胞に、CD3またはCD28遺伝子のいずれかを標的とするEdit-R All-in-one EGFPレンチウイルスsgRNAベクターを用いて、0.3 MOIで形質導入しました。all-in-oneレンチウイルス粒子は、hEF1αまたはmCMVプロモーターの下で構成的にCas9ヌクレアーゼを発現します。Non-targetingコントロール（NTC）ガイドを発現するレンチウイルス粒子をネガティブコントロールとして使用しました。レンチウイルスの形質導入と増殖の後、細胞をソーティングせず細胞集団（Pool）として分析するか、またはソーティングを行って、最も明るい（Top）または最も暗い（Dim）EGFP発現細胞集団を濃縮しました。次に、細胞のPool、Top、およびDimの集団をTIDE分析にかけ、ゲノム編集の割合を評価しました。

Edit-Rの 2ベクターシステムとall-in-oneシステムの比較

Comparison of Edit-R two-vector and All-in-one systems

2ベクターシステムの場合、hEF1αまたはmCMVの制御下でCas9を構成的に発現するためのレンチウイルス粒子を細胞に0.3 MOIで形質導入し、10 µg/mLブラストサイジンで10日間選択した後、all-in-one Non-targetingコントロール（NTC）あるいは、DNMT3BまたはPPIBをターゲットとするsgRNAレンチウイルス粒子を0.3 MOIで形質導入しました。all-in-oneシステムでは、all-in-one Non-targetingコントロール（NTC）あるいは、DNMT3BまたはPPIBをターゲットとするall-in-oneレンチウイルスsgRNAを野生型細胞に0.3 MOI にて形質導入し、2 µg/mLピューロマイシンで3日間選択しました。次に、細胞を溶解し、T7EIを用いたDNAミスマッチ検出アッセイを使用してindel（挿入・欠失）を分析しました。

Edit-R all-in-one Lentiviral sgRNAのベクターマップ

Edit-R all-in-one Lentiviral sgRNAベクターバックボーンでは、遺伝子特異的ガイドRNAはヒトU6プロモーターの制御下で発現し、Cas9およびピューロマイシン耐性マーカー（Puro^R）またはEGFPマーカーの発現はヒトEF1αまたはマウスCMVプロモーターのいずれかから駆動されます。プラスミドには、大腸菌での増殖と選択のためのアンピシリン耐性マーカーが含まれています。

Vector Element	Utility
Cas9	Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with a sgRNA
T2A	Self-cleaving peptide allows for simultaneous expression of puromycin resistance and Cas9 protein from a single transcript
Puro^R	Puromycin resistance marker permits antibiotic selection of transduced mammalian cells
EGFP	Fluorescent marker permits FACS selection of transduced mammalian cells
mCMV	Mouse cytomegalovirus immediate early promoter
hEF1α	Human elongation factor 1 alpha short promoter
U6	Human RNA polymerase III promoter U6
sgRNA	Optimized single guide RNA, a fusion of gene-specific crRNA with the tracrRNA scaffold
5' LTR	5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genome
Ψ	Psi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems
RRE	Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes
WPRE	Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells
3' SIN LTR	3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles
SV40 pA	Simian virus 40 polyadenylation signal
pUC ori	pUC origin of replication
SV40 ori	Simian virus 40 origin of replication
Amp^R	Ampicillin resistance gene for vector propagation in E. coli cultures

Our neutral cutting controls target an inactive region of the genome to provide a quantitative measure of cutting efficiency by mismatch detection assays, while maintaining a negative or negligible phenotype.

The neutral cutting controls target safe harbor genomic loci: human AAVS1 located on chromosome 19 and mouse Rosa26 on chromosome 6. Safe harbor loci are well-characterized, widely accepted, inactive genomic regions.

How is the neutral cutting control different from the non-targeting controls we offer?

Our non-targeting control guide RNAs do not target any region in the genome. Non-targeting designs control for phenotypic changes that may be caused by the delivery of the CRISPR-Cas9 reagents in the absence of cutting. However, the non-targeting controls cannot indicate changes caused by Cas9 cutting because they do not result in cutting by the Cas9 enzyme.

Our positive controls target established housekeeping genes to offer insight into phenotypic changes that might be due to Cas9 cutting but are not specific to cutting within the gene of interest. However, there may be cell types or culture conditions under which cutting at the housekeeping gene locus could result in a phenotype, or experimental situations where disrupting a gene is not desired. To overcome this potentially confounding effect, the neutral cutting control can be included to provide more robust results and increased confidence in the phenotypes observed following editing of the target gene.

Neutral cutting controls can be helpful if there is concern that an observed phenotype is not gene-specific. In particular, neutral cutting controls are helpful as an additional control in screening applications and when a phenotype is observed with a positive control guide RNA. Additionally, this guide can be used in discovery settings where disruption of a gene could affect downstream phenotypes and results.